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J. Biol. Chem., Vol. 281, Issue 12, 8275-8285, March 24, 2006

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Variable Reactivity of an Engineered Cysteine at Position 338 in Cystic Fibrosis Transmembrane Conductance Regulator Reflects Different Chemical States of the Thiol^*

From the Department of Physiology and Pharmacology, Oregon Health & Science University, Portland, Oregon 97239

In a previous study of T338C CFTR (cystic fibrosis transmembrane conductance regulator) we found that protons and thiol-directed reagents modified channel properties in a manner consistent with the hypothesis that this residue lies within the conduction path, but the observed reactivity was not consistent with the presence of a single thiolate species in the pore. Here we report results consistent with the notion that the thiol moiety can exist in at least three chemical states, the simple thiol, and two altered states. One of the altered states displays reactivity toward thiols like dithiothreitol and 2-mercaptoethanol as well as reagents: mixed disulfides (methanethiosulfonate reagents: MTSET⁺, MTSES^-) and an alkylating agent (iodoacetamide). The other altered state is unreactive. The phenotype associated with the reactive, altered state could be replicated by exposing oocytes expressing T338C CFTR to CuCl₂, but not by glutathionylation or nitrosylation of the thiol or by oxidation with hydrogen peroxide. The results are consistent with the hypothesis that substituting a cysteine at 338 can create an adventitious metal binding site. Metal liganding alters thiol reactivity and may, in some cases, catalyze oxidation of the thiol to an unreactive form such as a sulfinic or sulfonic acid.

Received for publication, November 21, 2005 , and in revised form, January 23, 2006.

^* This work was supported in part by the National Institutes of Health, NIDDK Grant DK45880 and Cystic Fibrosis Foundation Grant DAWSON0210. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by National Institutes of Health NIDDK Grants DK60312 and DK070755.

² Supported by Cystic Fibrosis Foundation Grant SERRAN04F0.

³ To whom correspondence should be addressed. L334, 3181 S.W. Sam Jackson Park Rd., Portland, OR 97239. Tel.: 503-494-8262; Fax: 503-494-4352; E-mail: dawsonda{at}ohsu.edu.

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